Showing papers on "Linear epitope published in 2008"
TL;DR: Investigating the role of immunoglobulin isotype, epitope density, and antigen/antibody ratio on the capacity of immune complexes to activate the classical and alternative pathways of human complement helps to explain the pathogenesis of complement activation in various autoimmune and immune complex diseases such as systemic lupus erythematosus, autoimmune thyroiditis and others.
Abstract: A systematic study has been carried out to investigate the role of immunoglobulin isotype, epitope density, and antigen/antibody ratio on the capacity of immune complexes to activate the classical and alternative pathways of human complement and for the complexes subsequently to bind to erythrocyte C3b-C4b receptors (CRI). For this purpose, a series of chimaeric monoclonal anti-NIP antibodies was used, which all shared the same combining site but had different human constant domains. Antigen epitope density was varied by coupling different numbers of NIP hapten molecules to bovine serum albumin. All three parameters affect complement fixation. In general, complement activation is better in antibody excess and at equivalence than it is in antigen excess, and better at high epitope density than at low epitope density, although the effects are variable for different immunoglobulin isotypes and for the two pathways. It has been confirmed that IgG1 and IgG3 are good activators of the classical pathway and are tolerant to variations in both epitope density and antigen/antibody ratio. IgG4 and IgA do not activate the classical pathway in any circumstances. IgG2 activates the classical pathway only at high epitope density and at equivalence or antibody excess. IgM activates the classical pathway well only at the higher epitope densities and at equivalence or antibody excess but, in addition, shows an interesting and unexpected prozone phenomenon where immune complex in antibody excess inhibits complement activation by the classical pathway. The results of the alternative pathway activation are strikingly different. IgA is by far the best activator of the alternative pathway and is relatively tolerant to epitope density and to antigen/antibody ratio. IgM, IgG1 and IgG3 do not significantly activate the alternative pathway in any circumstances. IgG2 is the best IgG subclass for alternative pathway activation but requires high epitope density and equivalence or antibody excess. Binding to CR1 in general parallels the amount of complement fixed independent to the pathway by which it is fixed. However, IgG1 and IgG3 complexes in antigen excess activate complement well but bind poorly to CR1. Nascently formed complexes seem to bind complement in a way that is similar to that bound by preformed complexes, but are then less able to bind to red cell CR1. These observations help to explain the pathogenesis of complement activation in various autoimmune and immune complex diseases such as systemic lupus erythematosus, autoimmune thyroiditis and others.
197 citations
Patent•
30 Jul 2008TL;DR: In this paper, a multispecific epitope binding proteins, methods of making, and uses thereof in the prevention, management, treatment or diagnosis of acute or chronic diseases are discussed.
Abstract: The present invention relates to multispecific epitope binding proteins, methods of making, and uses thereof in the prevention, management, treatment or diagnosis of acute or chronic diseases.
186 citations
TL;DR: It is demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies and shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.
Abstract: We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.
115 citations
TL;DR: A strategy to introduce epitope tag–encoding DNA into endogenous loci by homologous recombination–mediated 'knock-in' is developed.
Abstract: We developed a strategy to introduce epitope tag–encoding DNA into endogenous loci by homologous recombination–mediated ‘knock-in’. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation–microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.
97 citations
TL;DR: No differences were observed in the ranking of the relative reactivities of five lysine residues, however, the N‐terminal amino group was found to be part of the discontinuous epitope.
Abstract: A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.
95 citations
TL;DR: The identification of a linear B-cell epitope at the N-terminus of the E2 protein by screening a phage-displayed random 12-peptide library with the neutralizing monoclonal antibody HQ06 directed against the E 2 protein is described.
84 citations
TL;DR: This analysis shows how polymorphism at sites throughout the HLA class I molecule can influence the interaction of the Bw4 epitope with KIR3DL1, and this influence is likely mediated by changes in the peptides bound, which alter the conformation ofThe Bw 4 epitope.
Abstract: KIR3DL1 is a polymorphic, inhibitory NK cell receptor specific for the Bw4 epitope carried by subsets of HLA-A and HLA-B allotypes. The Bw4 epitope of HLA-B*5101 and HLA-B*1513 is determined by the NIALR sequence motif at positions 77, 80, 81, 82, and 83 in the alpha(1) helix. Mutation of these positions to the residues present in the alternative and nonfunctional Bw6 motif showed that the functional activity of the Bw4 epitopes of B*5101 and B*1513 is retained after substitution at positions 77, 80, and 81, but lost after substitution of position 83. Mutation of leucine to arginine at position 82 led to loss of function for B*5101 but not for B*1513. Further mutagenesis, in which B*1513 residues were replaced by their B*5101 counterparts, showed that polymorphisms in all three extracellular domains contribute to this functional difference. Prominent were positions 67 in the alpha(1) domain, 116 in the alpha(2) domain, and 194 in the alpha(3) domain. Lesser contributions were made by additional positions in the alpha(2) domain. These positions are not part of the Bw4 epitope and include residues shaping the B and F pockets that determine the sequence and conformation of the peptides bound by HLA class I molecules. This analysis shows how polymorphism at sites throughout the HLA class I molecule can influence the interaction of the Bw4 epitope with KIR3DL1. This influence is likely mediated by changes in the peptides bound, which alter the conformation of the Bw4 epitope.
64 citations
TL;DR: It is shown that an effective vaccine must induce antibodies to both conserved and more invariant epitopes to minimize virus escape and highlight the challenges inherent in developing HCV vaccines.
Abstract: A challenge in hepatitis C virus (HCV) vaccine development is defining conserved protective epitopes. A cluster of these epitopes comprises an immunodominant domain on the E2 glycoprotein, designated domain B. CBH-2 is a neutralizing human monoclonal antibody to a domain B epitope that is highly conserved. Alanine scanning demonstrated that the epitope involves residues G523, G530, and D535 that are also contact residues for E2 binding to CD81, a coreceptor required for virus entry into cells. However, another residue, located at position 431 and thus at a considerable distance in the linear sequence of E2, also contributes to the CBH-2 epitope. A single amino acid substitution at this residue results in escape from CBH-2-mediated neutralization in a genotype 1a virus. These results highlight the challenges inherent in developing HCV vaccines and show that an effective vaccine must induce antibodies to both conserved and more invariant epitopes to minimize virus escape.
59 citations
TL;DR: The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence, and that Tn glycosylation at the central threonine within thePDTRP core epitope region shifts the conformational equilibrium away from the type I Beta- turn conformation and toward a more rigid and extended state.
Abstract: MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.
56 citations
TL;DR: It is demonstrated that the lentivirus lytic peptide-2 domain in the cytoplasmic tail affected the accessibility of the 4E10 epitope, as well as virus infectivity, and strongly suggested immune escape from antibody responses targeting this region.
Abstract: The broadly neutralizing monoclonal antibody (MAb) 4E10 recognizes a linear epitope in the C terminus of the membrane-proximal external region (MPER) of gp41. This epitope is particularly attractive for vaccine design because it is highly conserved among human immunodeficiency virus type 1 (HIV-1) strains and neutralization escape in vivo has not been observed. Multiple env genes were cloned from an HIV-1 subtype C virus isolated from a 7-year-old perinatally infected child who had anti-MPER neutralizing antibodies. One clone (TM20.13) was resistant to 4E10 neutralization as a result of an F673L substitution in the MPER. Frequency analysis showed that F673L was present in 33% of the viral variants and in all cases was linked to the presence of an intact 2F5 epitope. Two other envelope clones were sensitive to 4E10 neutralization, but TM20.5 was 10-fold less sensitive than TM20.6. Substitutions at positions 674 and 677 within the MPER rendered TM20.5 more sensitive to 4E10 but had no effect on TM20.6. Using chimeric and mutant constructs of these two variants, we further demonstrated that the lentivirus lytic peptide-2 domain in the cytoplasmic tail affected the accessibility of the 4E10 epitope, as well as virus infectivity. Collectively, these genetic changes in the face of a neutralizing antibody response to the MPER strongly suggested immune escape from antibody responses targeting this region.
49 citations
TL;DR: A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis.
TL;DR: Fifty-six Newcastle disease virus strains collected from 2000 to 2006 could be grouped into subgenotype VIId, but they displayed cumulative mutations in and around the linear epitope of hemagglutinin-neuraminidase with time.
Abstract: Fifty-six Newcastle disease virus strains collected from 2000 to 2006 could be grouped into subgenotype VIId. However, they displayed cumulative mutations in and around the linear epitope of hemagglutinin-neuraminidase (residues 345 to 353) with time. The antigenicities of the variants that became predominant in Korea differ from each other and from the wild type.
TL;DR: Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67, and suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity.
Abstract: Objective: To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow “molecular positioning” of epitope sites for B- and T-cell reactivity. Research Design and Methods: Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs. Results: The C-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the C-terminal domain. Inclusion of epitope sites in the PLP- and N-terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAb to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T cell sequences restricted by HLA DRB1*0401 were aligned to solvent exposed regions of the GAD65 structure and co-localized within the two B-cell epitope clusters. The continuous C-terminal epitope region of GAD65 was structurally highly flexible, so differing markedly from the equivalent region of GAD67. Conclusions: Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B- and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T cell reactivity.
TL;DR: The number and location of epitopes recognized by SLF sera using recombinant proteins encoded by the full‐length or overlapping subclones of this cDNA are determined and suggest that 11 amino acids are critical for the recognition of this molecule by these autoantibodies.
Abstract: Autoantibodies to Ro/SSA are commonly found in sera of patients with systemic lupus erythematosus (SLE) and primary Sjogren's syndrome. The presence of these antibodies is related to lymphopenia, photosensitive dermatitis, and pulmonary and renal disease, suggesting that they have an immunopathologic role [1-6]. We previously isolated a cDNA clone which encodes the 52-kD human Ro/SSA protein. In this study we have determined the number and location of epitopes recognized by SLE sera using recombinant proteins encoded by the full-length or overlapping subclones of this cDNA. An immunodominant epitope was detected using Western blots and ELISA on the NH2-terminal side of this protein's putative leucine zipper. The data suggest that 11 amino acids are critical for the recognition of this molecule by these autoantibodies. Although the titres of anti-52-kD Ro/SSA antibodies vary between different patient sera, no heterogeneity in the location of antigenic epitopes to which their autoantibodies bound was detected. This homogeneous pattern of reactivity to a single rather than multiple regions of this protein is unusual for lupus autoantigens which have been identified, and suggests that these antibodies may have arisen as by a cross-reaction to an epitope on another molecule.
TL;DR: A panel of monoclonal antibodies directed against the N-terminus region of the coat protein of strain PPV W was generated by immunizing mice with recombinant peptides, and results obtained in ELISA, Western blot, and TAS-MIA correlated with B-cell recognition prediction.
TL;DR: A peptide inhibitor of HIV‐1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120, since it neither replicates the oligo saccharide footprint on the antibody nor most of the contact residues.
Abstract: MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K(d)s ranging from 0.4 to 200 microM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 A resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man(9)GlcNAc(2) (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man(9)GlcNAc(2), and only three of the antibody residues that interact with Man(9)GlcNAc(2) also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.
TL;DR: The results showed that A21 recognized a conformational epitope comprising a large region mostly from ErbBB2 extracellular subdomain I with several surface‐exposed residues important for the binding affinity, which provided unique functional properties of A21 that are quite different from two broadly used anti‐ErbB2 mAbs, Herceptin and 2C4.
Abstract: Anti-ErbB2 antibodies targeting distinct epitopes can have different biological functions on cancer cells. A21 prepared by surface epitope masking (SEM) method is a tumor-inhibitory anti-ErbB2 monoclonal antibody. Previously we engineered a single chain chimeric antibody chA21 with potential for therapy of ErbB2-overexpressing tumors. Here, we mapped the A21 epitope on ErbB2 extracellular domain (ECD) by screening a combinatorial phage display peptide library, serial subdomain deletion, and mutagenesis scanning. X-ray crystal structure of the A21 scFv fragment at 2.1 A resolution was also determined. A molecular model of Ag-Ab complex was then constructed based on the crystal structures of the A21 scFv and ErbB2 ECD. Some of biological functions of the A21 mAb and its derivative antibodies including their tumor cell growth inhibition and effects on the expression, internalization, and phosphorylation of ErbB2 receptor were also investigated. The results showed that A21 recognized a conformational epitope comprising a large region mostly from ErbB2 extracellular subdomain I with several surface-exposed residues important for the binding affinity. These data provide unique functional properties of A21 that are quite different from two broadly used anti-ErbB2 mAbs, Herceptin and 2C4. It suggested that the A21 epitope may be another valuable target for designing new anti-ErbB2 therapeutics.
TL;DR: The results show that antigen structure plays a significant role in the shaping of the helper T‐cell response against HIV gp120 in humans, and may prove to be useful in the development of rationally designed vaccines against pathogens such as HIV.
Abstract: The development of an effective vaccine against HIV/AIDS has been hampered, in part, by a poor understanding of the rules governing helper T-cell epitope immunodominance. Studies in mice have shown that antigen structure modulates epitope immunodominance by affecting the processing and subsequent presentation of helper T-cell epitopes. Previous epitope mapping studies showed that the immunodominant helper T-cell epitopes in mice immunized with gp120 were found flanking flexible loops of the protein. In this report, we show that helper T-cell epitopes against gp120 in humans infected with HIV are also found flanking flexible loops. Immunodominant epitopes were found to be located primarily in the outer domain, an average of 12 residues C-terminal to flexible loops. In the less immunogenic inner domain, epitopes were found an average of five residues N-terminal to conserved regions of the protein, once again placing the epitopes C-terminal to flexible loops. These results show that antigen structure plays a significant role in the shaping of the helper T-cell response against HIV gp120 in humans. This relationship between antigen structure and helper T-cell epitope immunodominance may prove to be useful in the development of rationally designed vaccines against pathogens such as HIV.
TL;DR: The use of single epitope-specific antibodies to differentially block functions of NSP4 is a feasible approach to determine the functional domain structure of this important RV virulence factor.
TL;DR: It was shown that immunogenicity of the subdominant epitope M2(127-135) was enhanced when the anchor residues of the dominant epitope were mutated, suggesting that the immunodominant epitope induces a suppression of response to the sub Dominated epitope.
TL;DR: The results suggest that some individuals who carry the GPPAA sequence in their HLA‐DQ molecule recognize this epitope in Epstein‐Barr virus, which may have potential importance in EBV‐induced immune abnormalities, although cross‐reactivity against DQ molecules could not be demonstrated in the present study.
Abstract: A five amino acids-long sequence (GPPAA) in the region of the 57th amino acid of HLA-DQ8 beta chain, which seems to be important in defining the risk for type 1 diabetes, occurs also in the BERF4-encoded EBNA3C protein of Epstein-Barr virus (EBV) in six successive repeats. The antigenicity of this region was analysed using synthetic peptides containing different modifications of the GPPAA sequence. Two of the seven individuals who had acute EBV infection produced antibodies against an EBV-derived peptide (GPPAAGPPAAGPPAA) paralleling the EBNA2 antibodies. These two cases also contracted type 1 diabetes immediately after the infection. High antibody levels against this peptide were found in a total of 12% of EBV+ individuals, and in most cases antibodies remained at high levels for several years. Human sera as well as affinity-purified antibodies specific for the GPPAAGPPAAGPPAA peptide reacted also with shorter peptide analogues (GPPAAGPPAA and GPPAA), as well as with peptides containing the surrounding motifs from DQ8 beta chains. However, none of these antibodies bound to denatured DQ8 beta chains in immunoblotting. The charge of the 57th amino acid modulated the antigenicity of this epitope, as peptides from Asp-57-negative DQ molecules were reactive, while peptides from Asp-57-positive DQ molecules were not. The responsiveness was seen in both HLA-DQ8-positive and -negative subjects as well as in type 1 diabetic individuals. The results suggest that some individuals who carry the GPPAA sequence in their HLA-DQ molecule recognize this epitope in EBV. This phenomenon may have potential importance in EBV-induced immune abnormalities, although cross-reactivity against DQ molecules could not be demonstrated in the present study.
TL;DR: There remains an association between OspA(165-173) and antibiotic-refractory Lyme arthritis, and infection-induced autoimmunity is an attractive hypothesis to explain this outcome, however, molecular mimicry due to sequence homology between OSpA (165- 173) and a human peptide seems unlikely to be the critical mechanism.
TL;DR: It is concluded that phage‐display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.
Abstract: Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.
TL;DR: Observations support the idea that MPER epitopes on membranes are segmented in terms of how they are affected by envelope lipids, which may have implications for MPER‐based vaccine development.
TL;DR: The present study represents the first experimental mapping of a neutralization epitope of a polyomavirus family member and should be useful for clarifying the extent of cross-reactivity exhibited by the humoral immune response towards related primatepolyomaviruses.
TL;DR: This finding shows that AT20, as an immunogenic molecule, elicits broadly neutralizing anti-p17 antibodies, by recognizing a highly conserved conformational epitope within p17 functional epitope.
TL;DR: A computer algorithm that permits epitope prediction based on a collection of random peptide epitopes (mimotopes) isolated by antibody affinity purification is described, which demonstrates that the combination of these two prediction strategies provides a robust platform for epitope characterization.
TL;DR: It is shown that antibodies to MOMP can protect mice against a chlamydial infection in the presence or absence of T and B cells and was as effective in SCID as in WT C.B-17 mice.
Abstract: Monoclonal antibodies (MAbs) to the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) were characterized for their ability to neutralize the infectivity of this organism in vitro and in vivo. One of the MAbs (MoPn-23) recognizes a nonlinear epitope in the MOMP, MAb MoPn-40 binds to a linear epitope in the variable domain 1 (VD1), and MAb MoPn-32 recognizes the chlamydial lipopolysaccharide. MAb MoPn-23 neutralized 50% of the infectivity of Chlamydia, as measured in vitro by using HAK (FcIII) and HeLa-229 (FcIII) cells at a concentration 100 times lower than MAb MoPn-40. MAb MoPn-32 had no neutralizing ability. In comparison to the control normal mouse immunoglobulin G, passive immunization of BALB/c mice with MAb MoPn-23 resulted in a highly significant protection against an intranasal (i.n.) challenge as determined by the change in body weight, the weight of the lungs, and the yield of Chlamydia inclusion-forming units (IFU) from the lungs. Passive immunization with MAb MoPn-40 resulted in a lower degree of protection, and MAb MoPn-32 afforded no protection. MAb MoPn-23 was also tested for its ability to protect wild-type (WT) and severe combined immunodeficient (SCID) C.B-17 mice against an i.n. challenge. Protection based on total body weight, lung weight, and yield of Chlamydia IFU was as effective in SCID as in WT C.B-17 mice. In conclusion, antibodies to MOMP can protect mice against a chlamydial infection in the presence or absence of T and B cells.
TL;DR: Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.
Abstract: CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-γ) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-γ by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-γ and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.
TL;DR: It is demonstrated that compromised SP22 gene expression is sensitive and correlated with fertility, and this applies to ejaculated sperm as well as epididymal sperm, and a combinatorial targeting strategy may be beneficial.